- Email: [email protected]
Separation of isoenzymes of citrate synthase and isocitrate dehydrogenase by fast protein liquid chromatography
FEBS 3380
Volume 196, number 2
February
1986
Separation of isoenzymes of citrate synthase and isocitrate dehydrogenase by fast protein liquid chromatography C.G. Mitchell, S. O’Neil, H.C. Reeves and P.D.J. Weitzman* Department of Biochemistry,
University of Bath, Bath BA2 7A Y, England
Received 9 December 1985 Fast protein liquid chromatography (FPLC) has been shown to be a rapid and effective method of separating isoenzymes of citrate synthase and isocitrate dehydrogenase in extracts of Pseudomonas aeruginosa and Acinetobacter calcoaceticus. The advantages of FPLC over conventional methods of fractionation are discussed and it is suggested that this may be a valuable and more general technique for isoenzyme resolution. FPLC
Isoenzyme separation
Citrate synthase
Isocitrate dehydrogenase
(Pseudomonas aeruginosa, Acinetobacter calcoaceticus)
allosteric regulatory properties not exhibited by the small isoenzymes. A rapid, reliable and highrecovery method for separating these isoenzyme pairs would be a valuable tool in the study both of their occurrence over a wide range of organisms and of any variations in the relative proportions of the isoenzymes under different physiological conditions [ 11. We have therefore investigated the use of FPLC for the separation of the isoenzymes of citrate synthase and isocitrate dehydrogenase. Expectations that this method of isoenzyme resolution might be effective, rapid and operable with small amounts of material were clearly fulfilled. The technique may find more general application in both the analytical and preparative separation of isoenzymes.
1. INTRODUCTION A variety employed for zymes, e.g. matography, These methods
of experimental methods may be the analytical separation of isoengel filtration, ion-exchange chrocentrifugation and electrophoresis.
may also be used for preparativescale separations but suffer from the disadvantage of being relatively slow procedures. The introduction by Pharmacia of an apparatus system for ‘fast protein liquid chromatography’ (FPLC) offers the opportunity to perform extremely rapid chromatography on newly developed ion-exchange materials with very high resolution. This technique should, in principle, be advantageously applicable to the resolution of mixtures of isoenzymes. In the course of our studies on enzymes of the citric acid cycle in diverse bacteria, two isoenzymic forms of citrate synthase (EC 4.1.3.7) have been discovered in various species of Pseudomonas [ 1,2] and two isoenzymes of isocitrate dehydrogenase (EC 1.1 .1.42) have been shown to be present in Acinetobacter calcoaceticus [3-51. In both these cases, the isoenzymes differ very substantially in molecular mass, the large forms displaying
2. EXPERIMENTAL 2.1. Organisms and preparation of cell extracts Pseudomonas aeruginosa, strain 8602, was kindly provided by Professor P.H. Clarke (University College, London). A. calcoaceticus, strain 4B, has been described [4] and was originally isolated from water by P.D.J.W. Ps. aeruginosa was grown in
* To whom correspondence should be addressed Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/86/$3.50
0
1986 Federation
of European
Biochemical
Societies
211
Volume 196, number 2
FEBSLETTERS
200 ml of nutrient broth (Oxoid) in a 500-ml flask shaking at 37°C for 16 h. The cells were harvested by centrifugation at 15000 x g for 10 min, resuspended in 4 ml of 20 mM Tris-HCI, pH 7.0, containing 1 mM EDTA and 0.1 M KQ, and sonicated (with cooling on ice) for a total of 2 min (4 x 30 s bursts with 30 s cooling between each). After centrifugation at 15000 x g for 10 min the supernatant was treated with 2% (w/v) aqueous solution of protamine sulphate (1 mg per 10 mg of extract protein) and stirred on ice for 10 min. The mixture was then centrifuged at 15000 x g for 20 min and the supernatant retained (protein content approx. 10 mg/ml). A. calcoaceticus was grown in the same way as above, but at 32°C. After harvesting, the cells were suspended in 2 ml of 20 mM Tris-HCl, pH 8.0, containing IO mM MgClz and 1 mM EDTA; sonication and treatment with protamine sulphate were carried out as above to give a final supernatant containing protein at 6 mg/ml. 2.2. Fast profein liquid c~romatogra~~~ FPLC was carried out using the equipment supplied by Pharmacia (see [6]). In the case of Ps. aeruginosa, the prepared extract was diluted approx. lo-fold with 20 mM Tris-HCI, pH 7.0, containing 1 mM EDTA to give a final protein concentration of 1 mg/ml. This was filtered through a 0.45 pm Millipore filter and 10 ml were applied to a Mono Q (HR 515) anion-exchange column (1 ml bed volume) equilibrated with 20 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane* HCI, pH 7.0. The column was washed with 4 column volumes (4 ml) of this buffer and elution was then continued using a gradient of O-O.2 M KC1 in the same buffer at a flow rate of 1 ml/min.. The column effluent was monitored for protein at 280 nm; citrate synthase was assayed as in 171. In the case of A. calcoaceficus, 500 ~1 of the prepared extract were applied to the Mono Q column equilibrated with 20 mM triethanol~ine-HCl, pH 7.7, and the column was washed with 4 ml of the same buffer. Protein elution was then achieved with a gradient of O-O.35 M NaCl in the same buffer at a flow rate of 2 ml/min. Effluent protein was monitored at 280 nm and isocitrate dehydrogenase was assayed as in [8]. All FPLC runs were conducted at room temperature and the fractions obtained were stored on ice. 212
February 1986
3. RESULTS AND DISCUSSION Two research projects in this laboratory have been concerned with isoenzymes in the citric acid cycle. Earlier studies on citrate synthase from a wide range of organisms (review 191) showed that this enzyme exists in a ‘small’ form (M, -100000) in Gram-positive bacteria and eukaryotes, and a ‘large’ form (f%fr -250000) in Gram-negative bacteria. Only the large enzyme displays regulatory sensitivity towards NADH and AMP 191.Solomon and Weitzman [l] reported the unusual occurrence of both small and large forms of citrate synthase in a mutant of Ps. aeruginosa and found that the relative proportions of the two forms varied with the stage of growth of the bacterial culture. In other work [3,4] we have shown that two isoenzymes of NADP-linked isocitrate dehydrogenase occur in A. ca~coacetic~sand a few other bacteria. One form has an M, of -300000 and is sensitive to several allosteric effecters [5,10]; the smaller isoenzyme has an M, of - 100000 and is not subject to these allosteric controls. The protein elution profiles obtained by FPLC of extracts of Ps. aerugi~osa and A. ca~coacetic~s are shown in figs 1 and 2, respectively. The small and large isoenzymes of citrate synthase are particularly well separated. This excellent resolution is the more remarkable if contrasted with the elution pattern on conventional ion-exchange chromatography on DEAE-Sephacel, when only a single peak of citrate synthase was observed. Fractionation by FPLC is therefore superior. Although the separation of A. calcoaceticus isocitrate dehydrogenase isoenzymes by FPLC (fig.2) is not as dramatic as that of the citrate synthases, the two isoenzymes are nevertheless clearly resolved. An important feature of both these elution profiles is their reproducibility, the isoenzymes being eluted in identical positions on repeat runs. A further important feature is the high recovery of enzyme activity on FPLC; recoveries in excess of 85% were obtained with both citrate synthase and isocitrate dehydrogenase, whereas recoveries from conventional chromatography on DEAE-Sephacel were in the region of 50-60%. Previous studies on these two isoenzyme systems have relied on classical methods of separation, particularly those exploiting the differences in molecular size, i.e. gel filtration [4,7] and zonal
February 1986
FEBS LETTERS
Volume 196, number 2
0.5
0.25
_--OF
_---
_--I 5
I 10
I 15
I 20
I 25
0
Elution volume (ml)
Fig. 1. FPLC separation of isoenzymes of citrate synthase from Ps. ueruginosu. Experimental conditions are described in the text. S and L refer to the small and large isoenzymes.
centrifugation [3]. These two techniques, as well as conventional ion-exchange chromatography, are relatively time-consuming; FPLC is considerably faster. In the experiments reported here, once the salt gradient had started, elution of the isoenzymes was complete within lo-20 min; the whole FPLC procedure took -30 min, in contrast to the several hours generally taken by gel filtration or DEAESephacel chromatography. Because of the high speed of FPLC, all experiments could be done at room temperature without significant loss of enzyme activity, thereby avoiding the inconvenience of cold-room operations. The speed of FPLC also reduces the risk of problems arising from the presence of proteases in cell extracts. Separation by gel filtration generally results in several-fold dilution of eluted enzymes, whereas the FPLC fractionation resulted in the elution of all the isoenzymes in very small volumes. One advantage of this is that relatively smaller amounts of enzyme need to be applied in FPLC in order to measure accurately the eluted activity. Another benefit is that the presence of the eluted enzyme in high concentration and with high recovery permits
further examination, e.g. gel electrophoresis, to be undertaken without prior additional steps to concentrate the eluted products. The results presented here clearly demonstrate the effectiveness of FPLC in achieving rapid and convenient separation of small amounts of isoenzymes in very high yield. We have made further use of these findings in exploring the incidence of citrate synthase isoenzymes in a range of Pseudomonas species [2] and in examining the roles of the two isoenzymes of Acinetobacter isocitrate dehydrogenase during adaptation of this organism to growth on acetate ([ll]; Reeves, H.C. et al., to be published). Although there have been a few other reports of isoenzyme separation by FPLC [12-141 the technique merits greater awareness and attention. The results presented here, together with recent work from this laboratory on the separation of glutamine synthetase isoforms [ 151, suggest that the technique of FPLC may find wide application to the separation, on both analytical and preparative scales, of a variety of isoenzyme systems.
213
Volume 196, number 2
FEBS LETTERS
February
r
1986
0.16
Or
I
I
5
10
15
20
Elution votume fmlt
Fig.2. FPLC separation of isoenzymes of isocitrate dehydrogenase from A. calcoaceticus. Experimental conditions are described in the text. S and L refer to the small and large &enzymes.
ACKNOWLEDGEMENTS This work was conducted while H.C.R. was on sabbatical leave from Arizona State University, and was supported in part by a grant from the National Science Foundation (PCM-7921714). Additional support was provided by grants (to P.D.J.W.) from the Science and Engineering Research Council (GR/A/82857 and GR/C/ 34377) and a Scientific Investigations Grant from The Royal Society to purchase the FPLC equipment .
REFERENCES (11 Solomon, M. and Weitzman, P.D.J. (1383) FEBS Lett. 155, 157-160. [2] Mitchell, C.G. and Weitzman, P.D.J. (1986) .I. Gen. Microbial., in press. [3] Self, C.H. and Weitzman, P.D.J. (1970) Nature 225, 644-645.
214
[it] Self, C.H. and Weitzman, P.D.J. (1972) Biochem. J. 130, 211-219, [5] Self, C.H., Parker, M.G. and Weitzman, P.D.J. (1973) Biochem. J. 132, 215-221. 161 ‘FPLC Ion-Exchange and Chromatofocusing’ (1985) Pharmacia Publication. [7] Weitzman, P.D.J. and Dunmore, P. (1%9) Biochim. Biophys. Acta 171, 198-200. 181 Garnak, M. and Reeves, H.C. (1979) J. Biol. Chem. 254, 7915-7920. [9] Weitzman, P.D.J. (1981) Adv. Microb. Physiol. 22, 185-244. IlO] Parker, M.G. and Weitzman, P.D.J. (1970) FEBS Lett. 7, 324-326. iI l] Reeves, H.C., @Neil, S. and Weitzman, P.D. J. (1983) FEBS Lett. 163, 265-268. (121 Haff, L.A., Fagerstam, L.G. and Barry, A.R. (1983) J. Chromatogr. 266, 409-425. 1131 Pressey, R. (1984) Eur. .I. Biochem. 144, 217-221. [14] Lindstedt, S. and Nordin, I. (1984) Biochem. J. 223, 119-127. [Is] Acaster, M.A. and Weitzman, P.D.J. (1985) FEBS Lett. 189, 241-244.
Volume 196, number 2
February
1986
Separation of isoenzymes of citrate synthase and isocitrate dehydrogenase by fast protein liquid chromatography C.G. Mitchell, S. O’Neil, H.C. Reeves and P.D.J. Weitzman* Department of Biochemistry,
University of Bath, Bath BA2 7A Y, England
Received 9 December 1985 Fast protein liquid chromatography (FPLC) has been shown to be a rapid and effective method of separating isoenzymes of citrate synthase and isocitrate dehydrogenase in extracts of Pseudomonas aeruginosa and Acinetobacter calcoaceticus. The advantages of FPLC over conventional methods of fractionation are discussed and it is suggested that this may be a valuable and more general technique for isoenzyme resolution. FPLC
Isoenzyme separation
Citrate synthase
Isocitrate dehydrogenase
(Pseudomonas aeruginosa, Acinetobacter calcoaceticus)
allosteric regulatory properties not exhibited by the small isoenzymes. A rapid, reliable and highrecovery method for separating these isoenzyme pairs would be a valuable tool in the study both of their occurrence over a wide range of organisms and of any variations in the relative proportions of the isoenzymes under different physiological conditions [ 11. We have therefore investigated the use of FPLC for the separation of the isoenzymes of citrate synthase and isocitrate dehydrogenase. Expectations that this method of isoenzyme resolution might be effective, rapid and operable with small amounts of material were clearly fulfilled. The technique may find more general application in both the analytical and preparative separation of isoenzymes.
1. INTRODUCTION A variety employed for zymes, e.g. matography, These methods
of experimental methods may be the analytical separation of isoengel filtration, ion-exchange chrocentrifugation and electrophoresis.
may also be used for preparativescale separations but suffer from the disadvantage of being relatively slow procedures. The introduction by Pharmacia of an apparatus system for ‘fast protein liquid chromatography’ (FPLC) offers the opportunity to perform extremely rapid chromatography on newly developed ion-exchange materials with very high resolution. This technique should, in principle, be advantageously applicable to the resolution of mixtures of isoenzymes. In the course of our studies on enzymes of the citric acid cycle in diverse bacteria, two isoenzymic forms of citrate synthase (EC 4.1.3.7) have been discovered in various species of Pseudomonas [ 1,2] and two isoenzymes of isocitrate dehydrogenase (EC 1.1 .1.42) have been shown to be present in Acinetobacter calcoaceticus [3-51. In both these cases, the isoenzymes differ very substantially in molecular mass, the large forms displaying
2. EXPERIMENTAL 2.1. Organisms and preparation of cell extracts Pseudomonas aeruginosa, strain 8602, was kindly provided by Professor P.H. Clarke (University College, London). A. calcoaceticus, strain 4B, has been described [4] and was originally isolated from water by P.D.J.W. Ps. aeruginosa was grown in
* To whom correspondence should be addressed Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/86/$3.50
0
1986 Federation
of European
Biochemical
Societies
211
Volume 196, number 2
FEBSLETTERS
200 ml of nutrient broth (Oxoid) in a 500-ml flask shaking at 37°C for 16 h. The cells were harvested by centrifugation at 15000 x g for 10 min, resuspended in 4 ml of 20 mM Tris-HCI, pH 7.0, containing 1 mM EDTA and 0.1 M KQ, and sonicated (with cooling on ice) for a total of 2 min (4 x 30 s bursts with 30 s cooling between each). After centrifugation at 15000 x g for 10 min the supernatant was treated with 2% (w/v) aqueous solution of protamine sulphate (1 mg per 10 mg of extract protein) and stirred on ice for 10 min. The mixture was then centrifuged at 15000 x g for 20 min and the supernatant retained (protein content approx. 10 mg/ml). A. calcoaceticus was grown in the same way as above, but at 32°C. After harvesting, the cells were suspended in 2 ml of 20 mM Tris-HCl, pH 8.0, containing IO mM MgClz and 1 mM EDTA; sonication and treatment with protamine sulphate were carried out as above to give a final supernatant containing protein at 6 mg/ml. 2.2. Fast profein liquid c~romatogra~~~ FPLC was carried out using the equipment supplied by Pharmacia (see [6]). In the case of Ps. aeruginosa, the prepared extract was diluted approx. lo-fold with 20 mM Tris-HCI, pH 7.0, containing 1 mM EDTA to give a final protein concentration of 1 mg/ml. This was filtered through a 0.45 pm Millipore filter and 10 ml were applied to a Mono Q (HR 515) anion-exchange column (1 ml bed volume) equilibrated with 20 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane* HCI, pH 7.0. The column was washed with 4 column volumes (4 ml) of this buffer and elution was then continued using a gradient of O-O.2 M KC1 in the same buffer at a flow rate of 1 ml/min.. The column effluent was monitored for protein at 280 nm; citrate synthase was assayed as in 171. In the case of A. calcoaceficus, 500 ~1 of the prepared extract were applied to the Mono Q column equilibrated with 20 mM triethanol~ine-HCl, pH 7.7, and the column was washed with 4 ml of the same buffer. Protein elution was then achieved with a gradient of O-O.35 M NaCl in the same buffer at a flow rate of 2 ml/min. Effluent protein was monitored at 280 nm and isocitrate dehydrogenase was assayed as in [8]. All FPLC runs were conducted at room temperature and the fractions obtained were stored on ice. 212
February 1986
3. RESULTS AND DISCUSSION Two research projects in this laboratory have been concerned with isoenzymes in the citric acid cycle. Earlier studies on citrate synthase from a wide range of organisms (review 191) showed that this enzyme exists in a ‘small’ form (M, -100000) in Gram-positive bacteria and eukaryotes, and a ‘large’ form (f%fr -250000) in Gram-negative bacteria. Only the large enzyme displays regulatory sensitivity towards NADH and AMP 191.Solomon and Weitzman [l] reported the unusual occurrence of both small and large forms of citrate synthase in a mutant of Ps. aeruginosa and found that the relative proportions of the two forms varied with the stage of growth of the bacterial culture. In other work [3,4] we have shown that two isoenzymes of NADP-linked isocitrate dehydrogenase occur in A. ca~coacetic~sand a few other bacteria. One form has an M, of -300000 and is sensitive to several allosteric effecters [5,10]; the smaller isoenzyme has an M, of - 100000 and is not subject to these allosteric controls. The protein elution profiles obtained by FPLC of extracts of Ps. aerugi~osa and A. ca~coacetic~s are shown in figs 1 and 2, respectively. The small and large isoenzymes of citrate synthase are particularly well separated. This excellent resolution is the more remarkable if contrasted with the elution pattern on conventional ion-exchange chromatography on DEAE-Sephacel, when only a single peak of citrate synthase was observed. Fractionation by FPLC is therefore superior. Although the separation of A. calcoaceticus isocitrate dehydrogenase isoenzymes by FPLC (fig.2) is not as dramatic as that of the citrate synthases, the two isoenzymes are nevertheless clearly resolved. An important feature of both these elution profiles is their reproducibility, the isoenzymes being eluted in identical positions on repeat runs. A further important feature is the high recovery of enzyme activity on FPLC; recoveries in excess of 85% were obtained with both citrate synthase and isocitrate dehydrogenase, whereas recoveries from conventional chromatography on DEAE-Sephacel were in the region of 50-60%. Previous studies on these two isoenzyme systems have relied on classical methods of separation, particularly those exploiting the differences in molecular size, i.e. gel filtration [4,7] and zonal
February 1986
FEBS LETTERS
Volume 196, number 2
0.5
0.25
_--OF
_---
_--I 5
I 10
I 15
I 20
I 25
0
Elution volume (ml)
Fig. 1. FPLC separation of isoenzymes of citrate synthase from Ps. ueruginosu. Experimental conditions are described in the text. S and L refer to the small and large isoenzymes.
centrifugation [3]. These two techniques, as well as conventional ion-exchange chromatography, are relatively time-consuming; FPLC is considerably faster. In the experiments reported here, once the salt gradient had started, elution of the isoenzymes was complete within lo-20 min; the whole FPLC procedure took -30 min, in contrast to the several hours generally taken by gel filtration or DEAESephacel chromatography. Because of the high speed of FPLC, all experiments could be done at room temperature without significant loss of enzyme activity, thereby avoiding the inconvenience of cold-room operations. The speed of FPLC also reduces the risk of problems arising from the presence of proteases in cell extracts. Separation by gel filtration generally results in several-fold dilution of eluted enzymes, whereas the FPLC fractionation resulted in the elution of all the isoenzymes in very small volumes. One advantage of this is that relatively smaller amounts of enzyme need to be applied in FPLC in order to measure accurately the eluted activity. Another benefit is that the presence of the eluted enzyme in high concentration and with high recovery permits
further examination, e.g. gel electrophoresis, to be undertaken without prior additional steps to concentrate the eluted products. The results presented here clearly demonstrate the effectiveness of FPLC in achieving rapid and convenient separation of small amounts of isoenzymes in very high yield. We have made further use of these findings in exploring the incidence of citrate synthase isoenzymes in a range of Pseudomonas species [2] and in examining the roles of the two isoenzymes of Acinetobacter isocitrate dehydrogenase during adaptation of this organism to growth on acetate ([ll]; Reeves, H.C. et al., to be published). Although there have been a few other reports of isoenzyme separation by FPLC [12-141 the technique merits greater awareness and attention. The results presented here, together with recent work from this laboratory on the separation of glutamine synthetase isoforms [ 151, suggest that the technique of FPLC may find wide application to the separation, on both analytical and preparative scales, of a variety of isoenzyme systems.
213
Volume 196, number 2
FEBS LETTERS
February
r
1986
0.16
Or
I
I
5
10
15
20
Elution votume fmlt
Fig.2. FPLC separation of isoenzymes of isocitrate dehydrogenase from A. calcoaceticus. Experimental conditions are described in the text. S and L refer to the small and large &enzymes.
ACKNOWLEDGEMENTS This work was conducted while H.C.R. was on sabbatical leave from Arizona State University, and was supported in part by a grant from the National Science Foundation (PCM-7921714). Additional support was provided by grants (to P.D.J.W.) from the Science and Engineering Research Council (GR/A/82857 and GR/C/ 34377) and a Scientific Investigations Grant from The Royal Society to purchase the FPLC equipment .
REFERENCES (11 Solomon, M. and Weitzman, P.D.J. (1383) FEBS Lett. 155, 157-160. [2] Mitchell, C.G. and Weitzman, P.D.J. (1986) .I. Gen. Microbial., in press. [3] Self, C.H. and Weitzman, P.D.J. (1970) Nature 225, 644-645.
214
[it] Self, C.H. and Weitzman, P.D.J. (1972) Biochem. J. 130, 211-219, [5] Self, C.H., Parker, M.G. and Weitzman, P.D.J. (1973) Biochem. J. 132, 215-221. 161 ‘FPLC Ion-Exchange and Chromatofocusing’ (1985) Pharmacia Publication. [7] Weitzman, P.D.J. and Dunmore, P. (1%9) Biochim. Biophys. Acta 171, 198-200. 181 Garnak, M. and Reeves, H.C. (1979) J. Biol. Chem. 254, 7915-7920. [9] Weitzman, P.D.J. (1981) Adv. Microb. Physiol. 22, 185-244. IlO] Parker, M.G. and Weitzman, P.D.J. (1970) FEBS Lett. 7, 324-326. iI l] Reeves, H.C., @Neil, S. and Weitzman, P.D. J. (1983) FEBS Lett. 163, 265-268. (121 Haff, L.A., Fagerstam, L.G. and Barry, A.R. (1983) J. Chromatogr. 266, 409-425. 1131 Pressey, R. (1984) Eur. .I. Biochem. 144, 217-221. [14] Lindstedt, S. and Nordin, I. (1984) Biochem. J. 223, 119-127. [Is] Acaster, M.A. and Weitzman, P.D.J. (1985) FEBS Lett. 189, 241-244.